Enzyme deactivation denaturation

In vitro enzymatic polymerizations have the potential for processes that are more regio-selective and stereoselective, proceed under more moderate conditions, and are more benign toward the environment than the traditional chemical processes. However, little of this potential has been realized. A major problem is that the reaction rates are slow compared to non-enzymatic processes. Enzymatic polymerizations are limited to moderate temperatures (often no higher than 50-75°C) because enzymes are denaturated and deactivated at higher temperatures. Also, the effective concentrations of enzymes in many systems are low because the enzymes are not soluble. Research efforts to address these factors include enzyme immobilization to increase enzyme stability and activity, solubilization of enzymes by association with a surfactant or covalent bonding with an appropriate compound, and genetic engineering of enzymes to tailor their catalytic activity to specific applications. 

In order to improve this reaction, a proper understanding of all parameters affecting product yield is desired. Clearly, the high enzyme consumption is a major obstacle for an efficient and economically feasible process. A likely cause of the inefficient use of DERA in this conversion is enzyme deactivation resulting from a reaction of the substrates and (by-) products with the enzyme. In general, aldehydes and (z-halo carbonyls tend to denature enzymes because of irreversible reactions with amino acid residues, especially lysine residues. From the three-dimensional structure it is known that DERA contains several solvent-accessible lysine residues [25]. Moreover, the complicated reaction profile as shown in Scheme 6.5 indicates the potential pitfalls of this reaction. 

Immobilization has other advantages it can slow enzyme deactivation by inhibiting protease attack and minimizing shear, interfacial, temperature, or solvent denaturation. As for the scarcity of some potentially very useful enzymes, it may be only a temporary problem. The development of cloning techniques, and probably the very increase in demand will result in lower prices. One spectacular instance is sialyl aldolase (see Table I). Industrial production of this enzyme by the gene-cloned strain of Escherichia coli has been reported.1,2 Sialylaldolase is now available from Toyobo at a moderate price. 

Although very interesting biotranformations have been reported in supercritical carbon dioxide, this solvent has been found to affect enzyme activity adversely. CO can react reversibly with free amino groups (lysine residues, specifically) on the surface of the protein to form carbamates, leading to low activity enzyme. [21]. Furthermore, carbon dioxide dissolves in water at molar concentrations at moderate pressures (<100 bar) and rapidly forms H COj. This can create some problems in biocatalytic reactions because many enzymes are denatured (unfolded and/or deactivated) at low pH. Enzymes can also be denatured by pressurization/depressuriza-tion cycles. For all of them, it is necessary to develop new enzyme stabilization strategies. 

Other mechanisms for enzyme denaturation in the presence of surfactants have also been proposed. One hypothesis is that the high charge densities of ionic surfactants increase the probability of them binding strongly to protein sites. This causes conformational changes of the enzyme which subsequently leads to further enzyme deactivation [99,103],... 

Effect of Temperature and pH. The temperature dependence of enzymes often follows the rule that a 10°C increase in temperature doubles the activity. However, this is only tme as long as the enzyme is not deactivated by the thermal denaturation characteristic for enzymes and other proteins. The three-dimensional stmcture of an enzyme molecule, which is vital for the activity of the molecule, is governed by many forces and interactions such as hydrogen bonding, hydrophobic interactions, and van der Waals forces. At low temperatures the molecule is constrained by these forces as the temperature increases, the thermal motion of the various regions of the enzyme increases until finally the molecule is no longer able to maintain its stmcture or its activity. Most enzymes have temperature optima between 40 and 60°C. However, thermostable enzymes exist with optima near 100°C. 

When the rate of an enzyme catalyzed reaction is studied as a function of temperature, it is found that the rate passes through a maximum. The existence of an optimum temperature can be explained by considering the effect of temperature on the catalytic reaction itself and on the enzyme denaturation reaction. In the low temperature range (around room temperature) there is little denaturation, and increasing the temperature increases the rate of the catalytic reaction in the usual manner. As the temperature rises, deactivation arising from protein denaturation becomes more and more important, so the observed overall rate eventually will begin to fall off. At temperatures in excess of 50 to 60 °C, most enzymes are completely denatured, and the observed rates are essentially zero. 

Enzyme activity generally passes through a maximum as the pH of the system in question is varied. However, the optimum pH varies with substrate concentration and temperature. Provided that the pH is not changed too far from the optimum value corresponding to the maximum rate, the changes of rate with pH are reversible and reproducible. However, if the solutions are made too acid or too alkaline, the activity of the enzyme may be irreversibly destroyed. Irreversible deactivation is usually attributed to denaturation of the proteinaceous enzyme. The range of pH in which reversible behavior is observed is generally small and this... 

Piper and Fenton [10] indicated that extreme acidity or basicity of the gastric juice denaturalize the enzymatic activity of the pepsin, which shows has a higher activity at a pH = 2. At pH = 5 the enzyme starts to deactivate and at pH= 7, the enzyme irreversibly lose its activity. Fig. 3 shows the pepsin UV-visible spectra before and after interaction with the zeolites while Fig 4 shows the enzymatic activity of the denatured hemoglobin proteolysis versus reaction time. 

Most ultrasonic experiments are carried out in temperature controlled systems to ensure that isothermal conditions are maintained. Even a small general increase in microbial temperature can influence both the active and passive transport systems of the cell membrane/wall and this in turn may lead to an increased uptake of compounds. If the temperature is not controlled then sonication could result in a large temperature increase which will lead to the denaturation (deactivation) of enzymes, proteins and other cellular components present within the microorganism [7]. 

The environmental tolerance of enzymes closely parallels other substances associated with live processes. They tolerate a relatively narrow temperature span and with denaturation (deactivation) occurring at temperatures generally above 50 C (122 F). Greatly reduced activity usually occurs well above the freezing point of water. Enzymes have a low tolerance to a pH below 4.0, and a minimal to no tolerance of certain organic solvents (alcohol, acetone, etc.), and destruction by numerous organic and inorganic substances. 

There are two types of inhibitors. Reversible inhibitors bind to an enzyme in a reversible fashion and can be removed by dialysis (or dilution) to restore full enzyme activity. Irreversible inhibitors cannot be removed by dialysis and, in effect, permanently deactivate or denature the enzyme. 

Although the interest of scientists in peroxidase enzymes has increased tremendously during the past decades, the application of these enzymes as biocatalysts in industrial processes is still negligible. Often the low activity and the fragile nature of these enzymes make their use challenging and sometimes results in poor productivities. Different aspects including heme deactivation (Chap. 12), redox potential modulation (Chap. 4), protein denaturation, and substrate availability have to be dealt with. 

The definition of a more efficient enzymatic system could be based on the separation of the catalytic cycle of the enzyme and the degradation step by the Mn3+ reactive species in MnP systems. The Mn3+-chelates present several advantages in their use as oxidants. They are more tolerant to protein denaturing conditions such as extremes of temperature, pH, oxidants, organic solvents, detergents, and proteases, and they are smaller than proteins therefore, they can penetrate microporous barriers inaccessible to proteins. The optimization of the production of the Mn3+-chelate will have to be compatible with the minimal consumption and deactivation of the enzyme. 

Enzymes in the presence of water can attack and break apart proteins and biological body waste products and dirt. Why is it advised not to boil the washing The enzyme is a protein and can be deactivated or denatured if heated too strongly. 

In spite of a long-time paradigm that enzymes can be active only in their natural aqueous media and other solvents cause deactivation and denaturation of proteins, at present a growing number of investigations are devoted to enzymatic reactions in organic solvents (Klibanov, 2001 Ke et al., 1996 Koskinen and Klibanov, 1996 and references therein). Such enzymes as a-chymotrypsin, subtilisin ribonuclease, pancreatuc lipase, and horse radish peroxidase have been found to be markedly active in organic solvents (alcohols, amines, tiols,anhydrous alkanes, acetonitril, dichloromethane, methyl acetate, etc.). 

Figure 32 includes results illustrating the performance of lipase/car-bon monolith systems in an acylation reaction. For comparison, the free lipase and a commercial immobilized lipase (Novozyme) were also tested. As expected, in all cases the specific activity of immobilized lipase was foimd to be lower than that of the free enzyme. Such a difference is usually ascribed to conformational changes of the enzyme, steric effects, or denaturation. For the monolithic biocatalysts, the activity of the immobilized catalyst relative to that of the pure enzyme was found to be 30-35%, and for the Novozyme catalyst about 80% in the first rim. However, the Novozyme catalyst underwent significant deactivation, in contrast to the carbon monolith-supported catalysts. The deactivation of the Novozyme catalyst in consecutive runs is probably a consequence of the instability of the support matrix under reaction conditions (101,102). 

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